The Contractor shall develop and implement a highly multiplexed targeted sequencing assay designed to detect toxicity during compound screening. The RASL-Seq HTS assay will monitor gene expression across 184 target genes and 20 housekeepi ng genes, in a format amenable to high throughput screening (HTS) that is cost effective and easy to use. Phase I will characterize assay performance using a set of 10 reference compounds and selected compounds from the National Toxicology Program (NTP), using HepG2 cells in a 384- well plate context. We will demonstrate assay utility using an HTS protocol that can test up to 10,000 compound treatments per day, while providing quantitative, precision dose response data. In Phase II, we will expand this assay to the RASL-Seq HTS Tox Profiler assay, which will include the -1100 toxicity-related genes exhibiting known expression changes, and a surrogate whole genome pathway and mechanism assay of 1,000 to 3,000 genes. We will establish whether a 1536-well format can be used, and will design an automated process that can test 100,000 compounds per day.